Antibacterial photosensitization through activation of coproporphyrinogen oxidase.

Gram-positive bacteria cause the majority of skin and soft tissue infections (SSTIs), resulting in the most common reason for clinic visits in the United States. Recently, it was discovered that Gram-positive pathogens use a unique heme biosynthesis pathway, which implicates this pathway as a target for development of antibacterial therapies. We report here the identification of a small-molecule activator of coproporphyrinogen oxidase (CgoX) from Gram-positive bacteria, an enzyme essential for heme biosynthesis. Activation of CgoX induces accumulation of coproporphyrin III and leads to photosensitization of Gram-positive pathogens. In combination with light, CgoX activation reduces bacterial burden in murine models of SSTI. Thus, small-molecule activation of CgoX represents an effective strategy for the development of light-based antimicrobial therapies.

Gametophyte Development Needs Mitochondrial Coproporphyrinogen III Oxidase Function.

Tetrapyrrole biosynthesis is one of the most essential metabolic pathways in almost all organisms. Coproporphyrinogen III oxidase (CPO) catalyzes the conversion of coproporphyrinogen III into protoporphyrinogen IX in this pathway. Here, we report that mutation in the Arabidopsis (Arabidopsis thaliana) CPO-coding gene At5g63290 (AtHEMN1) adversely affects silique length, ovule number, and seed set. Athemn1 mutant alleles were transmitted via both male and female gametes, but homozygous mutants were never recovered. Plants carrying Athemn1 mutant alleles showed defects in gametophyte development, including nonviable pollen and embryo sacs with unfused polar nuclei. Improper differentiation of the central cell led to defects in endosperm development. Consequently, embryo development was arrested at the globular stage. The mutant phenotype was completely rescued by transgenic expression of AtHEMN1 Promoter and transcript analyses indicated that AtHEMN1 is expressed mainly in floral tissues and developing seeds. AtHEMN1-green fluorescent protein fusion protein was found targeted to mitochondria. Loss of AtHEMN1 function increased coproporphyrinogen III level and reduced protoporphyrinogen IX level, suggesting the impairment of tetrapyrrole biosynthesis. Blockage of tetrapyrrole biosynthesis in the AtHEMN1 mutant led to increased reactive oxygen species (ROS) accumulation in anthers and embryo sacs, as evidenced by nitroblue tetrazolium staining. Our results suggest that the accumulated ROS disrupts mitochondrial function by altering their membrane polarity in floral tissues. This study highlights the role of mitochondrial ROS homeostasis in gametophyte and seed development and sheds new light on tetrapyrrole/heme biosynthesis in plant mitochondria.

The role of coproporphyrinogen III oxidase and ferrochelatase genes in heme biosynthesis and regulation in Aspergillus niger.

Heme is a suggested limiting factor in peroxidase production by Aspergillus spp., which are well-known suitable hosts for heterologous protein production. In this study, the role of genes coding for coproporphyrinogen III oxidase (hemF) and ferrochelatase (hemH) was analyzed by means of deletion and overexpression to obtain more insight in fungal heme biosynthesis and regulation. These enzymes represent steps in the heme biosynthetic pathway downstream of the siroheme branch and are suggested to play a role in regulation of the pathway. Based on genome mining, both enzymes deviate in cellular localization and protein domain structure from their Saccharomyces cerevisiae counterparts. The lethal phenotype of deletion of hemF or hemH could be remediated by heme supplementation confirming that Aspergillus niger is capable of hemin uptake. Nevertheless, both gene deletion mutants showed an extremely impaired growth even with hemin supplementation which could be slightly improved by media modifications and the use of hemoglobin as heme source. The hyphae of the mutant strains displayed pinkish coloration and red autofluorescence under UV indicative of cellular porphyrin accumulation. HPLC analysis confirmed accumulation of specific porphyrins, thereby confirming the function of the two proteins in heme biosynthesis. Overexpression of hemH, but not hemF or the aminolevulinic acid synthase encoding hemA, modestly increased the cellular heme content, which was apparently insufficient to increase activity of endogenous peroxidase and cytochrome P450 enzyme activities. Overexpression of all three genes increased the cellular accumulation of porphyrin intermediates suggesting regulatory mechanisms operating in the final steps of the fungal heme biosynthesis pathway.

Dual gene defects involving delta-aminolaevulinate dehydratase and coproporphyrinogen oxidase in a porphyria patient.

Summary A Caucasian male had symptoms of acute porphyria, with increases in urinary delta-aminolaevulinic acid (ALA), porphobilinogen (PBG) and coproporphyrin that were consistent with hereditary coproporphyria (HCP). However, a greater than expected increase in ALA, compared with PBG, and a substantial increase in erythrocyte zinc protoporphyrin, suggested additional ALA dehydratase (ALAD) deficiency. Nucleotide sequence analysis of coproporphyrinogen oxidase (CPO) cDNA of the patient, but not of the parents, revealed a novel nucleotide transition G835-->C, resulting in an amino acid change, G279R. The mutant CPO protein expressed in Escherichia coli was unstable, and produced about 5% of activity compared with the wild-type CPO. Erythrocyte ALAD activity was 32% of normal in the proband. Nucleotide sequence analysis of cloned ALAD cDNAs from the patient revealed a C36-->G base transition (F12L amino acid change). The F12L ALAD mutation, which was found in the mother and a brother, was previously described, and is known to lack any enzyme activity. This patient thus represents the first case of porphyria where both CPO and ALAD deficiencies were demonstrated at the molecular level.

Alcohol and porphyrin metabolism.

Alcohol is a porphyrinogenic agent which may cause disturbances in porphyrin metabolism in healthy persons as well as biochemical and clinical manifestations of acute and chronic hepatic porphyrias. After excessive consumption of alcohol, a temporary, clinically asymptomatic secondary hepatic coproporphyrinuria is observable, which can become persistent in cases of alcohol-induced liver damage. Nowadays, the alcohol-liver-porphyrinuria syndrome is the first to be mentioned in secondary hepatic disturbances of porphyrin metabolism. Acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria and hereditary coproporphyria) are considered to be molecular regulatory diseases, in contrast to non-acute, chronic hepatic porphyria, clinically appearing as porphyria cutanea tarda (PCT). Porphyrins do not accumulate in the liver in acute porphyrias, whereas in chronic hepatic porphyrias they do. Thus, chronic hepatic porphyria is a porphyrin-accumulation disease, whereas acute hepatic porphyrias are haem-pathway-dysregulation diseases, characterized in general by induction of delta-aminolevulinic acid synthase in the liver and excessive stimulation of the pathway without storage of porphyrins in the liver. The clinical expression of acute hepatic porphyrias can be triggered by alcohol, because alcohol augments the inducibility of delta-aminolevulinic acid synthase. In chronic hepatic porphyrias, however, which are already associated with liver damage, alcohol potentiates the disturbance of the decarboxylation of uro- and heptacarboxyporphyrinogen, which is followed by a hepatic accumulation of uro- and heptacarboxyporphyrin and their sometimes extreme urinary excretion. Especially in persons with a genetic deficiency of uroporphyrinogen decarboxylase, but also in patients with the so-called sporadic variety of PCT, alcohol is able to transform an asymptomatic coproporphyrinuria into PCT. Alcohol has many biochemical and clinical effects on porphyrin and haem synthesis both in humans and laboratory animals. Ethanol suppresses the activity of porphobilinogen synthase (synonym: delta-aminolevulinic acid dehydratase), uroporphyrinogen decarboxylase, coproporphyrinogen oxidase and ferrochelatase, whereas it induces the first and rate-limiting enzyme in the pathway, delta-aminolevulinic acid synthase and also porphobilinogen deaminase. Therefore, teetotalism is a therapeutically and prophylactically important measure in all types of hepatic porphyrias.

Coproporphyrin excretion by Azorhizobium caulinodans under micro-aerobic conditions.

Azorhizobium caulinodans ORS571 was found to excrete moderate amounts of a fluorescent pigment into the culture medium in response to dissolved oxygen tensions below 1.0 kPa. The pigment was identified as coproporphyrin, on the basis of its optical and fluorescence spectra. FixLJ and fixK mutant derivatives of ORS571 were found to excrete 25-fold higher amounts of coproporphyrin under micro-aerobic conditions than the wild type strain. These observations suggest that A. caulinodans switches from an aerobic to an anaerobic coproporphyrinogen oxidase when the dissolved oxygen tension falls below 1.0 kPa and that the fixLJ and fixK genes are involved in the regulation of expression of the anaerobic coproporphyrinogen oxidase.

Investigation of the subcellular location of the tetrapyrrole-biosynthesis enzyme coproporphyrinogen oxidase in higher plants.

The subcellular location of two enzymes in the biosynthetic pathway for protoporphyrin IX, coproporphyrinogen (coprogen) oxidase (EC 1.3.3.3) and protoporphyrinogen (protogen) oxidase (EC 1.3.3.4) has been investigated in etiolated pea (Pisum sativum) leaves and spadices of cuckoo-pint (Arum maculatum). Plant tissue homogenized in isotonic buffer was subjected to subcellular fractionation to prepare mitochondria and plastids essentially free of contamination by other cellular organelles, as determined by marker enzymes. Protogen oxidase activity measured fluorimetrically was reproducibly found in both mitochondria and etioplasts. In contrast, coprogen oxidase could be detected only in etioplasts, using either a coupled fluorimetric assay or a sensitive radiochemical method. The implications of these results for the synthesis of mitochondrial haem in plants is discussed.

Protoporphyrin formation in Rhizobium japonicum.

The obligately aerobic soybean root nodule bacterium Rhizobium japonicum produces large amounts of heme (iron protoporphyrin) only under low oxygen tensions, such as exist in the symbiotic root nodule. Aerobically incubated suspensions of both laboratory-cultured and symbiotic bacteria (bacteroids) metabolize delta-aminolevulinic acid to uroporphyrin, coproporphyrin, and protoporphyrin. Under anaerobic conditions, suspensions of laboratory-cultured bacteria form greatly reduced amounts of protoporphyrin from delta-aminolevulinic acid, whereas protoporphyrin formation by bacteroid suspensions is unaffected by anaerobiosis, suggesting that bacteroids form protoporphyrin under anaerobic conditions more readily than do free-living bacteria. Oxygen is the major terminal electron acceptor for coproporphyrinogen oxidation in cell-free extracts of both bacteroids and free-living bacteria. In the absence of oxygen, ATP, NADP, Mg2+, and L-methionine are required for protoporphyrin formation in vitro. In the presence of these supplements, coproporphyrinogenase activity under anaerobic conditions is 5 to 10% of that observed under aerobic conditions. Two mechanisms for coproporphyrinogen oxidation exist in R. japonicum: an oxygen-dependent process and an anaerobic oxidation in which electrons are transferred to NADP. The significance of these findings with regard to heme biosynthesis in the microaerophilic soybean root nodule is discussed.